应用FIA型微生物传感器测定谷氨酸含量的研究

目前应用酶或微生物细胞作为分子识别元件构建生物传感器测定谷氨酸含量的研究引起了广泛的兴趣,并陆续有实例报道。在这些报道中人们依据不同的酶催反应选择相应的离子选择性电极,如CO₂电极、NH₄⁺电极等,而测量对象有直接的测定谷氨酸,也有测定谷氨酸单钠的间接测定法。本文选用了可固定大量细胞的固定化细胞柱与流动注入法相结合的测量方法,并根据细胞柱的动力学模型分析动态响应曲线,计算测量结果,提高了测量精度,扩大了测量范围。     

Variability of human immunodeficiency virus type 1 group O strains isolated from Cameroonian patients living in France.

Human immunodeficiency virus type 1 (HIV-1) nucleotide sequences encoding p24Gag and the Env C2V3 region were obtained from seven patients who were selected on the basis of having paradoxical seronegativity on a subset of HIV enzyme-linked immunosorbent assay detection kits and having atypical Western blot (immunoblot) reactivity. Sequence analyses showed that all of these strains were more closely related to the recently described Cameroonian HIV isolates of group O (HIV-1 outlier) than to group M (HIV-1 major). All seven patients had Cameroonian origins but were living in France at the time the blood samples were taken. Characterization of a large number of group M strains has to date revealed eight distinct genetic subtypes (A to H). Genetic distances between sequences from available group O isolates were generally comparable to those observed in M intersubtype sequence comparisons, showing that the group O viruses are genetically very diverse. Analysis of sequences from these seven new viral strains, combined with the three previously characterized group O strains, revealed few discernable phylogenetic clustering patterns among the 10 patients' viral sequences. The level of diversity among group O sequences suggests that they may have a comparable (or greater) age than the M group sequences, although for unknown reasons, the latter group dispersed first and is the dominant lineage in the pandemic.     

Effects of nucleotide analogs on ATP hydrolysis by P-type ATPases. Comparison between the canine kidney (Na++ K+) ATPase and maize root H+-ATPase

The mechanism by which chemical energy is converted into an electrochemical gradient by P-type ATPase is not completely understood. The effects of ATP analogs on the canine kidney (Na⁺+ K⁺) ATPase were compared to effects of the same analogs on the maize (Zea mays L. cv. W7551) root H⁺-ATPase in order to identify probes for the ATP binding site of the maize root enzyme and to determine potential similarities of ATP hydrolysis mechanisms in these two enzymes. Six compounds able to modify the ATP binding site covalently were compared. These compounds could be classed into three distinct groups based on activity. The first group had little or no effect on catalytic activity of either enzyme and included 7-chloro-4-nitrobenz-2-oxa-1.3-diazole. The second group, which included azido adenine analogs. fluorescein isothiocyanate and 5′-p-fluorosulfonylbenzoyladenine, were inhibitors of ATP hydrolysis by both enzymes. However, the sensitivity of the (Na⁺+ K⁺) ATPase to inhibition was much greater than that exhibited by the maize root enzyme. The third group, which included periodate treated nucleotide derivatives and 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate. inhibited both enzymes similarly. This initial screening of these covalent modifiers indicated that 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate was the optimal covalent modifier of the ATP binding site of the maize root enzyme. Certain reagents were much more effective against the (Na⁺+ K⁺) ATPase than the maize root enzyme, possibly indicating differences in the ATP binding and hydrolysis pathway for these two enzymes. Two ATP analogs that are not covalent modifiers were also tested: the trinitrophenyl derivatives of adenine nucleotides were better than 5′-adenylylimidodiphosphate for use as an ATP binding probe.     

REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae.

Protein phosphatase type 1 (PP1) is encoded by GLC7, an essential gene in Saccharomyces cerevisiae. The GLC7 phosphatase is required for glucose repression and appears to function antagonistically to the SNF1 protein kinase. Previously, we characterized a mutation, glc7-T152K, that relieves glucose repression but does not interfere with the function of GLC7 in glycogen metabolism. We proposed that the mutant GLC7T152K phosphatase is defective in its interaction with a regulatory subunit that directs participation of PP1 in the glucose repression mechanism. Here, we present evidence that REG1, a protein required for glucose repression, is one such regulatory subunit. We show that REG1 is physically associated with GLC7. REG1 interacts with GLC7 strongly and specifically in the two-hybrid system, and REG1 and GLC7 fusion proteins co-immunoprecipitate from cell extracts. Moreover, overexpression of a REG1 fusion protein suppresses the glc7-T152K mutant defect in glucose repression. This and other genetic evidence indicate that the two proteins function together in regulating glucose repression. These results suggest that REG1 is a regulatory subunit of PP1 that targets its activity to proteins in the glucose repression regulatory pathway.     

Physicochemical properties of alkaline serine proteases in alcohol

The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1g/ml and that of alcalase was 48.1g/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters,l-Ala-l-Ala-(d-orl-)Pro-l-Phe-OMe andl-Ala-l-Ala-(d-orl-)Ala-l-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.     

On the production,elemental composition (C,N, P) and distribution of photosynthetic organic matter in the Southern Black Sea

Chemical oceanographic understanding of the southernBlack Sea has been improved by recent measurements ofthe optical transparency, phytoplankton biomass (interms of chlorophyll-a and particulate organic matter)and primary productivity. During the spring-autmunperiod of 1995–1996, light generally penetrated onlyinto the upper 15–40 m, with an attenuation coefficientvarying between 0.125 and 0.350 m^2122;1. The averagechlorophyll-a (Chl-a) concentrations for the euphoticzone ranged from 0.1 to 1.5 μg l^2122;1. Coherentsub-surface Chl-a maxima were formed near the base ofthe euphotic zone only in summer. Production rate variedbetween 247 and 1925 in the spring and between 405 and687 mgC m^2122;2 d^2122;1 in the summer-autumn period.The average POM concentrations in the euphotic zonevaried regionally and seasonally between 3.8 and28.6 μm for POC, 0.5 and 3.1 μm for PON and0.02 and 0.1 μm for PP. Atomic ratios of C/N, C/Pand N/P, derived from the regressions of POM data,ranged between 7.5 and 9.6, 109 and 165, and 11.2 and16.6, respectively. In the suboxic/anoxic interface,the elemental ratios change substantially due to anaccumulation of PP cohering to Fe and Mn oxides. Thechemocline boundaries and the distinct chemicalfeatures of the oxic/anoxic transition layer (the so-called suboxic zone) are all located at specificdensity surfaces; however, they exhibit remarkablespatial and temporal variations both in their positionand in their magnitude, which permit the definition of long-term changes in the biochemical properties of theBlack Sea upper layer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Elemental composition of seston and nutrient dynamics in the Sea of Marmara

The Sea of Marmara, an intercontinental basin with shallow and narrowstraits, connects the Black and Mediterranean Seas. Data obtained during1991–1996 have permitted the determination of the elementalcomposition of seston in the euphotic zone and the N:P ratio of thesubhalocline waters of the Marmara Sea. Since primary production is alwayslimited to the less saline upper layer (15–20 m), of the Marmara Sea,the subhalocline waters of Mediteranean origin are always rich in nutrients(NO₃ + NO₂ = 8–10 μm, PO₄ = 0.8–1.2 μm) but depleted in dissolvedoxygen (30–50 μm) throughout the basin, yielding an -O_{_2} : N : P ratio of 178 : 9 : 1. Pollution of the surfacewaters since the 60s has modified the subhalocline nutrient chemistryslightly. In the euphotic zone, the N : P ratio of the seston changes from5.9 to 9.5 between the less and more productive periods. Though the biologyof the Marmara has changed significantly during the previous two decades,the close relationship observed between the elemental composition of thesurface seston and the NO₃ : PO₄ ratio of thesubhalocline waters strongly suggests that during the whole year primaryproduction throughout the basin and POM export to the lower layer remainnitrogen-limited. This suggestion needs to be confirmed by bio-assays,biological studies and sediment trap data from the upper subhaloclinedepths. Nonetheless, the counterflows in the Marmara basin possessrelatively low N : P ratios in both dissolved and particulate nutrients andextend as far as the adjacent seas. This revised version was published online in July 2006 with corrections to the Cover Date.     

Detection of human blood by cloth-based enzyme immunoassay of immunoglobulin G

A specific and sensitive assay for the detection of human blood was developed using polyester cloth coated with goat anti-human IgG antibody to capture human IgG, an abundant and stable protein in blood. The captured IgG was detected by the reaction between goat anti-human IgG antibody-peroxidase conjugate and a chromogenic peroxidase substrate. Because the assay is simple and rapid, and permits simultaneous analysis of multiple samples, it has the potential to be used as a forensic test for human blood.     

Amine fluorescamine compounds inhibit oxidative phosphorylation in rat liver mitochondria

The reaction of fluorescamine with ammonia, benzylamine, o,p-dimethylbenzylamine, 2-phenylethylamine, p-aminobenzoic acid, and the mycosamine-containing macrolide antibiotic, amphotericin B, yield compounds which induce significant effects on mitochondrial activities. From their effects on energy-yielding processes which lead to transmembranous proton movements, the compounds may be divided into three classes. While all modifiers significantly inhibit proton movement induced by both ATP hydrolysis and electron transfer in mitochondria, their influence on the primary energy yielding steps are quite different. Class I modifiers, e.g., the compound made from amphotericin B, inhibit electron transfer but have no effect on the Pi release associated with ATP hydrolysis. Class II modifiers, e.g., the compound made from benzylamine, inhibit respiration but stimulate Pi release. Class III modifiers, e.g., the compound made from p-aminobenzoic acid, on the other hand, only slightly increase Pi release but have no effect on redox reactions. These and other effects of the modifiers are taken to mean that the proton movements and their associated energy-yielding processes are only linked indirectly. The effects of the modifiers on State 3 mitochondrial activities were also investigated. Although all the modifiers decrease the rates of both State 3 respiration and its coupled ATP synthesis, the efficiency of energy conversion measured by the P/O ratio remains unaltered.